Reproducible and scalable differentiation of highly pure cortical neurons from human induced pluripotent stem cells

Summary Human-induced-pluripotent-stem-cell (hiPSC)-derived neurons are valuable for investigating brain physiology and disease. Here, we present a protocol to differentiate hiPSCs into cortical neurons with high yield and purity. We describe neural induction via dual-SMAD inhibition, followed by spot-based differentiation to provide high quantities of neural precursors. We detail their enrichment, expansion, and purification to avoid unwanted cell fates and provide optimal conditions for neural rosette proliferation. These differentiated neurons are suitable for drug testing and co-culture studies. For complete details on the use and execution of this protocol, please refer to Paquet et al.1 and Weisheit et al..2


SUMMARY
Human-induced-pluripotent-stem-cell (hiPSC)-derived neurons are valuable for investigating brain physiology and disease. Here, we present a protocol to differentiate hiPSCs into cortical neurons with high yield and purity. We describe neural induction via dual-SMAD inhibition, followed by spot-based differentiation to provide high quantities of neural precursors. We detail their enrichment, expansion, and purification to avoid unwanted cell fates and provide optimal conditions for neural rosette proliferation. These differentiated neurons are suitable for drug testing and co-culture studies. For complete details on the use and execution of this protocol, please refer to Paquet et al. 1 and Weisheit et al.. 2

BEFORE YOU BEGIN
Institutional permissions Experiments on pluripotent stem cells need to be performed in accordance with relevant institutional and national guidelines and regulations and may need to be approved by an institutional review board (IRB) or similar committee. This was also performed for the experiments described in this study, where applicable.

Culture of human induced pluripotent stem cells (iPSCs)
Timing: around 1 week prior to starting the differentiation, with one iPSC maintenance split performed before starting the differentiation

Thawing of induced pluripotent stem cells (iPSCs)
a. Prepare vitronectin-coated plates: add 1 mL of vitronectin solution (1:100 in PBS) for each well of a 6-well plate, coat for at least 1 h at room temperature (RT -20 C-22 C) b. Prepare 9 mL DMEM-F12 and 2 mL E8 Flex/RI (RI = Rock-inhibitor Y27632, 10 mM) for each thawed tube, warm both to RT. c. Get stored cryovials with iPSCs from liquid nitrogen tank and thaw in a 37 C water bath until only a small ice clump remains.
2. Culture of iPSCs. a. iPSC lines growing in E8 Flex are typically split $1:6 every 3-4 days (e.g., Tuesday and Friday) and are kept in colonies. Before splitting, cells should form dense colonies with a smooth and uniform surface. b. Split cultures when (or preferentially before) the first of the following occurs: i. iPSC colonies become too dense (cells start to pile up) or too large.
ii. iPSC colonies show increased differentiation.
iii. iPSC colonies cover $80% of the surface area of the culture vessel, usually every 3-4 days. 3. Splitting iPSC colonies: a. Prepare and warm E8 Flex medium and DMEM-F12 to RT as needed, coat wells of a 6-well plate with vitronectin as needed b. Add 1.5 mL E8 Flex per well to recipient, vitronectin-coated plate (no RI is used for normal splitting).
Note: If iPSCs are split on a Friday, 3.5 mL can be added to skip feeding on the weekend.
c. If spontaneously differentiated cells are present, remove them from the plate using a dissection microscope, or by aspirating non-translucent areas/colonies on culture plates (see ''Critical'' paragraph below). d. Aspirate culture medium, wash cells with 1 mL PBS. e. Remove PBS, add 1 mL PBS with 0.5 mM EDTA and incubate 4-5 min at RT, monitor cells every 1-2 min to check morphology, cells are ready to split when colonies develop gaps in the middle but are still loosely attached. f. Aspirate PBS/EDTA without dislodging colonies. g. Forcefully add DMEM-F12 (half the volume of the split ratio, i.e., for a 1:6 split, add 3 mL) to the well using a sterile plastic Pasteur pipette to wash off colonies. This works best by pushing the DMEM-F12 'under the colonies' from the side. h. Pull up media, and push out forcefully to wash residual colonies off the plate, pipette up and down until small colonies remain (usually 2-5 times).
Note: iPSCs should always be kept in colonies to avoid cell death or spontaneous differentiation of single cells.
i. Avoid scraping the cells from the culture vessel during passaging, as differentiated cells preferentially stay attached. j. Quickly add 0.5 mL of cell suspension into each new well containing fresh E8 Flex media (prepared in step 3b) using the same Pasteur pipette before the clumps start to settle down in the pipette. k. Evenly distribute colonies by moving the plate back and forth in two directions. l. No feed is required for the next 2 days, do 2 mL feeds on following days until the next split.
Note: If using another iPSCs media (not E8 Flex) which does not contain heat-stabilized bFGF, timing of the feeds needs to be adjusted accordingly, i.e., feed every day or every other day.
CRITICAL: After thawing, iPSCs should be passaged at least once prior to starting the differentiation protocol. It is critical that aberrant differentiation of iPSCs is below 5% before the neural induction is started. If necessary, remove spontaneously differentiated cells from the iPSC cultures as per standard stem cell culture procedures (e.g., manual removal under a microscope or reduced incubation time of 4 min with PBS/EDTA, as iPSCs usually detach before differentiated cells). Note: All coating solutions are temperature sensitive and should therefore be aliquoted on ice, diluted in cold diluent and added to the respective plates as quickly as possible.

KEY RESOURCES
Alternatives: both Cultrex and Matrigel can be used as alternatives to GTX, according to individual manufacturer's instructions. Adjust the dilutions accordingly to achieve the same final concentrations.

Media for optional steps
To prepare L-ascorbic acid-2-phosphate stock solution (64 mg/mL), resuspend 500 mg in 7.81 mL sterile MilliQ water, sterile filter, aliquot and store at À20 C for up to one year.

Timing: 7 days
In this first step, neural induction from iPSCs is initiated using dual-SMAD inhibition based on previously published studies. [6][7][8] A schematic overview of this step is included in Figure 1A.
1. Prepare required number of iPSCs on vitronectin-coated 6-well plates as described above.
2. Prepare required number of GTX-coated wells of a 12-well plate; as a starting point coat two to three wells of a 12-well plate per well of a 6-well plate of iPSCs.
CRITICAL: Clean off spontaneously differentiated cells in the iPSC culture thoroughly prior to starting the neural induction, as these will inhibit neuronal differentiation. Aberrant differentiation of iPSCs should be <5%. Troubleshooting 1.
3. Split iPSCs into wells of a GTX-coated 12-well plate when they are 70%-80% confluent; this day is defined as Day-in-vitro 0 (DIV0). a. Add 0.5 mL Accutase to the well with iPSCs. b. Incubate 5-8 min at 37 C until most cells detach when shaking/tapping the plate; if unsure, monitor under a microscope that cells are detaching. c. Add 1 mL DMEM-F12, gently triturate to single cells with a P1000 pipette and count cells. d. Transfer required amount of cell suspension (1 million cells per well of a 12-well plate) into a 15 mL conical tube and spin 4 min at 170 g.
Note: All centrifugation steps are performed with these parameters at RT unless indicated otherwise.
e. Aspirate media and resuspend pellet to a single cell solution at 1 million cells/mL in NI supplemented with RI (10 mM). f. Plate 1 mL of cell suspension (1 million cells) per well of a 12-well plate on the prepared, GTXcoated plates (resulting in a cell density of 260,000 cells/cm 2 ). g. Evenly distribute cells by moving the plate back and forth in two directions.
Note: Cells should have formed a confluent monolayer on the next day ( Figure 1B). Increase plating density the next time if cells are not confluent on this day and decrease density if issues with detaching cells occur later.
Note: If required, cell clumps can be carefully removed using a vacuum pump, but plating density and resuspension to single cells should be optimized to avoid formation of clumps, also see troubleshooting 2.
4. Change NI media every day until DIV7.
a. The first days, feed 1 mL NI per well of a 12-well plate. b. Increase media volume to 1.5-2 mL on DIV5-7 to ensure sufficient supply with growth factors and nutrients.

Reagent Final concentration Amount
Can be stored at 4 C for up to 5 days.

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Note: Sufficient nutrient supply will improve the quality of the high-density spots in the next step.
Optional: use E6 -NI gradient for difficult-to-differentiate cell lines on DIV0 and DIV1 starting with the split; also see troubleshooting 3 for more information i. On DIV0, perform split as described above using 1 mL E6-NI gradient medium-1 instead of NI.
ii. On DIV1, change medium to 1 mL of E6-NI gradient medium-2. iii. On DIV2, change medium to NI and continue with the standard protocol.
Note: Depending on the cell density and iPSC line, each well of a 6-well plate of iPSCs will yield 2-4 million cells. We recommend optimizing this first split (starting cell density, number of cells per well of a 12-well plate) for each cell line depending on its proliferative capacity.

Neural rosette formation in spots
Timing: around 1 week In this step, neural precursor cells (NPCs) are plated in spots to achieve maximum cell density while allowing sufficient nutrient supply when fed once per day. High density plating is crucial to achieve high quality cortical neuron differentiations. A schematic overview of this step is included in Figure 1A.
5. On DIV6 (=one day before the split), coat wells of a 6-well plate with 1 mL of each PO and L as described under ''Materials and Equipment''. Depending on the proliferative capacity of the iPSC line, one full 12-well plate is usually split into 4-8 wells of a 6-well plate. 6. On the day of the split, the cells should look as depicted in Figure 1C Note: A crystalline sheet will appear, there should be no clear gaps in the sheet ( Figure 1D).
8. Perform the high density split into spots on DIV7, also see Methods video S1. a. Aspirate the medium and add 0.5 mL Accutase to the cells, incubate 10 min at 37 C. b. Add 1 mL DMEM-F12 (10 mM RI can be added to DMEM-F12 when more than one plate needs to be split simultaneously). c. Wash off cells with a P1000 pipette to obtain a single cell suspension, pool the suspension in a 15 mL conical tube and count the cells. d. Spin down cells, aspirate supernatant, resuspend cells in appropriate amount of NI/RI to get 30 million cells per mL, make sure to obtain a single cell suspension. e. Put one 250-350 mL drop per well on dried POL 6-well plates. The cell suspension should stay in the middle of the plate and not disperse (Methods video S1), do not introduce air bubbles. f. If necessary, spread the spot slightly by gently tilting and tapping the plate, such that its diameter is about 1/3 to 1/2 of the well diameter (depending on the spot volume).
Note: Avoid contact between the cell solution and the edges of the well, as this would pull cell solution towards the edge, resulting in deformed spots with lower cell density and uniformity.
g. Let cells attach for 60 min at RT without adding additional media; put the lid on the plate and keep inside the hood (i.e., do not put the cells into the incubator) to avoid dislodging the spot while cells are attaching. h. After 60 min, add 2 mL NI/RI very gently to the wall of the plate with a serological pipette, tilt the plate very gently to spread out the NI, ensure that cells stay in the spot and are not washed off ( Figure 1E). 9. The next day, shake or tap the plate to dislodge any non-attached/dead cells and replace the medium with fresh 2 mL NI; the spots should stay on the plate and form a dense, uniform cell layer ( Figures 1E and 1F). 10. Change NI media every day, 3-4 mL feeds are sufficient to bridge one weekend day. 11. On DIV10, switch to 2 mL NM media.

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12. Starting from DIV10, for subsequent feeds change NM media every day, 3-4 mL feeds are sufficient to bridge one weekend day. a. Around DIV10, a ''shoulder'' of partially non-cortical cells forms at the very edge of the spots (containing neural crest cells and other cells) while the high-density cells in the middle of the spots soon start forming neural rosettes ( Figure 1G).
CRITICAL: The quality of cells in the 12-well plate before making spots needs to be very high. The cells should be in a uniform layer and should not peel off the well or start dying (see Figures 1B and 1C) -if they do, the starting cell density in the 12-well plates needs to be optimized. For more proliferative iPSC lines, the day of making the spots can be shifted to DIV6 to ensure cells are confluent in the 12-well plate after plating but do not peel off on the day of making the spots. Troubleshooting 4 CRITICAL: Drying of the POL-coated plates is critical for the successful generation of spots. It is crucial to have an even crystalline sheet appear on the bottom of the plates, as spots will peel off if they are placed onto gaps in the sheet. The relative hydrophobicity of the sheet will contain the area covered by cells which allows for the high cell densities required for successful neuron differentiation. Troubleshooting 4 Optional: If not enough cells are available to prepare spots at the indicated density (30 million cells/mL) and volume (250-350 mL), spots can be made with smaller volumes but same density. This will however disproportionally decrease final rosette yield, as the area of the spot "shoulder" -containing potentially aberrantly differentiated cells -will be increased relative to the area of rosettes on the inside which are used for further differentiation (see Figure 2B and step 15 below).
Optional: If cell density is low on DIV10 (e.g., holes are forming) when changing to NM, bFGF (20 ng/mL) can be added to NM for the next 2 feeds. Troubleshooting 5 Note: On DIV13-15, the rosettes in the spots can be used to differentiate astrocytes using the serum-free protocol by Perriot et al. 9 Expansion of neural rosettes Timing: around 3 weeks In this step, the population of NPCs is expanded to increase the number of resulting neurons at the end of the differentiation protocol. To achieve this, it is crucial to maintain the integrity of neural rosettes. A schematic overview of this step is included in Figure 2A.
13. When neural rosettes appear, usually around DIV15 ( Figure 2B), feed cells for 2 consecutive days with 2 mL NM supplemented with bFGF (20 ng/mL) (= NM + bFGF), to support proliferation and expansion of the rosettes. 14. For subsequent feeds, change NM media every day, 3-4 mL feeds are sufficient to bridge one weekend day. 15. When rosettes start accumulating (usually around DIV20-23: the culture develops brownish, dense areas/circles visible under the microscope, Figure 2C), split cells with NRSR (STEMdiff Neural Rosette Selection Reagent), also illustrated in Methods video S2.
Note: NRSR is a reagent that is designed to specifically dislodge rosettes without breaking them apart, resulting in separation of rosettes from undesired cells. Here, it is used in combination with manual selection of neural rosettes inside the spot to increase the purity of the culture and enrich for NPCs; using this procedure, only one NRSR split is required to obtain pure neuron cultures.
a. Depending on the density and size of the spots, split the spots in a 3:2 to 3:4 ratio onto POLcoated 6-well plates; as a starting point split one spot into one well of a 6-well plate. b. Aspirate media. c. Add 1 mL NRSR, incubate 30-60 min at 37 C (optimize time for cell line used). d. Aspirate NRSR and add 1.5 mL NM. e. Manually isolate rosettes from inside the spot using a pipette tip (Methods video S2 and Figure 2C), do not touch the ''shoulder'' of the spot or cells outside the spot. f. Collect dislodged rosettes in a 15 mL conical tube, triturate slightly to break up larger clumps (5-103 with Pasteur pipette or P1000 if more careful), make sure to not dissociate the neural rosettes into single cells. g. Spin down cells, remove supernatant, carefully resuspend cells in NM + bFGF, triturate 1-23 with 5 mL serological pipette. h. Plate 2 mL of the cell suspension per well of a POL-coated 6-well plate.
Note: The goal of this step is to keep rosettes intact but break up aggregates of rosettes and give them more space for growth ( Figure 2D). Rosettes need to be grown at high density without generating too many single cells, as these will fall out of cell cycle, reducing final cell yield. Therefore, special attention should be paid when triturating the cell suspension. Troubleshooting 6 16. Feed with NM + bFGF on the next day.

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17. For subsequent feeds, change NM media (without bFGF) every day to every other day. 18. Cultures should contain single rosettes and small aggregates of rosettes, but few to no other cell types or large clumps of cells, the NPCs (radial-glia-like cells) in the middle of the rosettes will keep dividing and the cultures will become denser ( Figure 2D). 19. When rosettes become almost confluent (usually around DIV30-33), split again to give cells more space, also see Methods video S3. a. Split 3 wells into 3-5 wells, depending on density and proliferative capacity of the cell line. b. Incubate with 0.5 mL Accutase for 4 min at 37 C, do not aspirate, add 1 mL NM. c. Wash cells off with plastic Pasteur pipette or P1000 pipette. d. Pool and triturate gently with Pasteur pipette or P1000 to separate rosettes from each other; avoid making single cells. e. Spin cells down, resuspend in fresh NM + bFGF and plate in 2 mL per well of a 6-well plate. 20. Feed with NM + bFGF on the next day. 21. For subsequent feeds, feed with NM media every day to every other day.
Note: The cultures should look like the rosettes before the Accutase split, but less dense with more neurites ( Figure 2D). Troubleshooting 6

Freezing and thawing of neural rosettes
Timing: 15 min for each freezing or thawing procedure, 1 day to coat plates before thawing In this step, we describe how to freeze the neural rosettes for long-term storage in a liquid nitrogen tank, as mature cortical neurons cannot be frozen. We also describe how to thaw the neural rosettes. A schematic overview of this step is included in Figure 2A.
Pause point: Rosettes can be frozen at this point and stored in liquid nitrogen for extended periods of time (months to years). Long-term culture of the differentiated neurons can be resumed at any later timepoint, allowing for synchronization of different cell lines and cell types for long-term culture.
22. Freezing of neural rosettes works best between DIV35 and DIV41.
a. Add 0.5 mL Accutase to the cells, incubate for 4 min at 37 C. b. Do not aspirate Accutase, add 1 mL NM and triturate cells gently with Pasteur pipette without breaking up rosettes, collect cells in a 15 mL conical tube. c. Spin down cells and resuspend the NPCs in 1 mL cold Freeze Medium for Neural Rosettes for every 1-2 collected wells, depending on cell density; triturate 2-33 with 5 mL pipette. d. Transfer cell suspension to cryovials and freeze vials with 1 mL cell suspension in prechilled Mr. Frosty at -80 C, save a drop to genotype cells if needed. e. The next day, transfer frozen rosettes to liquid nitrogen tank (À80 C is not sufficient for longterm storage).
Optional: To facilitate planning, count cells before freezing by triturating a small, representative aliquot to single cells. Freeze down NPCs at about 10 million cells per cryovial to thaw them into one well of a 6-well plate. Document yield (i.e., counting the neurons obtained from the differentiation) and/or quality of the cells (e.g., by taking representative brightfield images) to optimize future differentiations and adjust subsequent experimental setups. there are larger clumps) and plate into one well of a 6-well plate (assuming cells were frozen at $10 million cells/vial), shake plate to distribute cells and transfer to incubator. 24. On day post thaw (DPT) 1, if the media is yellow, add 1-2 mL fresh NM + bFGF on top. 25. On DPT 2, do a full feed with 2 mL NM + bFGF. 26. Feed 2 mL NM on following days as needed. 27. On DPT 4-7, split the neurons for long-term culture as described below.
Note: Neuronal long-term cultures can also be generated directly from differentiated cells without freezing. In order to do so, perform another Accutase split as described in points 19-21 between DIV37-DIV41, and mature the rosettes further until DIV 43-48 when plating of the neurons for long-term cultures is performed (see below).
Note: as alternative to RVC, both RI or CEPT 10 can be added to thawing medium.
Optional: To ensure cortical identity of the differentiated neurons, stainings for neuronal markers can be performed as we describe in the Expected Outcomes section. If other neuronal cell types are detected, refer to troubleshooting 7.

Long-term culture of differentiated cortical neurons
Timing: 1 day to coat plates, 20-45 min for splitting the cells, 1 month to 1 year for longterm culture In this step, we describe how to plate the neurons in different well formats to ensure stable culture of the cells for up to 12 months.
Note: Cells should have recovered from thawing by rearranging into rosettes, with early neurons being generated as seen by neurite formation. The space between rosettes should be free of other cell types and dead cells that may be visible after thawing should be gone after media changes.
Note: Neuronal survival after the final split may vary. We recommend aiming for the highest possible density in the provided range that still allows uniform neuron growth without major clumping. If the density is too high, clumping will occur due to clustering of the neurons.
b. Prepare required number of POL-coated wells/plates as described above. c. Prepare the following before the split: i. one 15 mL tube with 4 mL NM per well of a 6-well plate with rosettes.
ii. required amounts of Accutase (0.5 mL per well of a 6-well plate with rosettes).
iii. the required amounts of plating medium: NB+/B27+ supplemented with DAPT (10 mM) and RVC (1:100); for plating: use 2 mL/ 1 mL / 0.5 mL for wells of 6-well/12-well/24-well plates respectively. d. Aspirate NM, add 0.5 mL Accutase to rosettes and incubate 6-10 min at 37 C (depending on density and morphology). Optional: Transfer single cell suspension through a 70 mm cell strainer into the prepared 15 mL tube to remove any remaining large clumps from the suspension. Large clumps should not remain in the suspension as they often contain proliferative NPCs and thus impair stable long-term neuronal culture. Pre-wet the cell strainer and the wall of the 15 mL tube with media to minimize cell loss and death.
h. Count cells (one well can contain 5-20 million cells depending on cell density). i. Transfer the required number of cells into a new tube, spin down. j. Aspirate supernatant, resuspend and gently triturate with P1000 pipette in 1 mL Plating medium until no clumps are visible anymore (usually 4-63). k. Add required media volume to reach recommended cell density and plate quickly (occasionally mix the tube when plating many wells).
Note: DAPT inhibits Notch signaling and thereby promotes terminal differentiation of NPCs into neurons.
Note: If neurons are to be plated onto glass coverslips for imaging, the coverslips need to be pre-treated with 30% HCl at least 12 h to roughen the glass surface. Afterwards, wash coverslips 4-53 with de-ionized water to fully remove the acid. Store coverslips in 100% Ethanol. Dry off Ethanol and coat the coverslips with POL as described above before use.
Note: Cells are very sensitive at this stage, therefore it is important to work as quickly as possible, minimize trituration, and to not introduce air bubbles. Always add cells directly into media if possible.
29. Extended culture of neurons over longer time periods (1 month-1 year). a. Feed the cells 2 days after plating with NB+/B27+ with DAPT. i. Before the feed, tap the plate to dislodge debris and dead cells into the supernatant. Note: 5-Fluorouracil is added to kill residual dividing cells, while Uridine is added to minimize the effect of 5-Fluorouracil on transcription and thus reduce toxicity in non-dividing cells.
c. Consecutively do half feeds with NB+/B27+ supplemented with DAPT and 5FU two to three times per week. i. DAPT should be added for a total of 7 days (starting with the day of plating).
ii. 5FU should be added for a total of 10-14 days (starting at the feed 3 days after plating).
Note: If a lot of cell death occurs during 5FU treatment, tap the plate to dislodge debris and do a full feed.
Note: Ensure that media volumes stay constant during half feeds by briefly tilting plates after removing media and checking contents in individual wells, media in peripheral wells may evaporate faster.
d. After the 5FU treatment ends, switch to half feeds with NB+/B27+ two to three times per week. e. Cells will start growing neurites the day after plating; after two weeks, the cells will have formed a dense neuritic network; there should be over 95% neurons and no large patches of other cell types, some astrocytes may be present 1-2 months after plating.
Note: We tested long-term neuron culture with different neuronal media. Neurons can also be fed with NB/B27, NB/B27 PLUS and will survive for long-term, but in our experience, the cultures are more stable in NB PLUS/B27 PLUS in terms of survival for extended culture periods of >4 months. We did not test Brainphys for long-term culture, because we noted increased contamination with astrocytes.

EXPECTED OUTCOMES
The presented protocol yields highly pure cultures of cortical forebrain neurons by ensuring constantly optimal differentiation conditions, e.g., uniformly high cell density, enrichment of neuronal rosettes, and removal of proliferative precursor cells. We tested our protocol in several different iPSC lines both before and after CRISPR/Cas9-mediated genomic edits. We can differentiate all tested iPSC lines robustly into cortical neurons, as shown in Figure 3A.
To ensure high quality of the differentiations and to confirm neuronal fate, we recommend performing quality control experiments, especially when establishing the differentiation of a new iPSC line. For this purpose, we describe the expected outcomes of the differentiation and include typical markers that are expressed during the differentiation. Additional quality control parameters that can be used to assess the generated neurons can be found in previously published studies. [6][7][8] In the first days of neuronal differentiation, the cells lose pluripotency markers such as Oct4 and NANOG and increase expression of neuroepithelial and cortical NPC markers such as Pax6, Nestin, and FoxG1 ( Figures 3B-3E). The NPCs proliferate and form neural rosettes by day 12-15, which are then purified and expanded. After the final split (around DIV41-47), the separated, single cells lose expression of neural progenitor markers such as PAX6 and Nestin and upregulate neuronal markers such as MAPT, MAP2, and NMDAR1 ( Figures 3E, 4A-4C). Overall, the combination of weekly splits as well as DAPT and 5FU treatment leads to a highly homogeneous population of neurons, by removing already postmitotic cells with extended processes during splits, and those still dividing after the final split by mitotic inhibition, respectively. Thus, terminal differentiation occurs within a narrow time frame, finally yielding a highly pure culture of cortical forebrain neurons.
Neurons differentiated from iPSCs using this protocol express typical markers such as MAP2, b3-Tubulin, and Tau directly after neuron differentiation both on mRNA and protein level ( Figures 4A  and 4C). Synapses are formed from the beginning and increase over time while the cultures mature further (Figures 4B and 4C). During this maturation, extensive neuritic networks are formed, neuronal markers are further upregulated, and post-mitotic cortical neurons dominate the culture while contaminating astrocytes are minimal ( Figures 4C-4E).
Mimicking layer formation in the human cortex, the protocol yields lower-layer, CTIP2-positive neurons and mid-layer, SATB2-positive neurons ( Figure 4F), with a larger number of deep-layer neurons. This is because deep layer neurons are formed first in human development with mid-and upper-layer neurons following afterward, which we block by DAPT and 5FU treatment. Performing these treatments earlier 11 or later 7 will alter the fraction of neurons from different layers. Cortical neurons are largely glutamatergic (vGLUT1-positive), with a small proportion of GAD67-positive GABAergic cells present ( Figure 4G).
We further confirmed electrophysiological excitatory activity of the neurons and demonstrated evoked action potentials and typical resting membrane potential. 1 Using Fluo4 time lapse Calcium

LIMITATIONS
In general, it is not possible to generate completely pure neuron cultures without any contaminating astrocytes. This is a well-known phenomenon, as both are derived from the same precursor cells. 8,12 By using DAPT/5FU treatment and ensuring that single neurons are plated, the number of contaminating astrocytes can however be limited to a minimum using the presented protocol.
Details of the protocol need to be adjusted to the iPSC line in use. Potentially, the starting cell number as well as split ratios during differentiation need to be increased or decreased to adjust for different proliferative behavior of different cell lines. In our experience, the vast majority of cell lines successfully differentiate into cortical neurons with the provided cell numbers.
Furthermore, drying of the POL plates for making spots depends on the humidity of the environment. We suspect that it might be more difficult to dry the plates in very humid environments (>70% humidity). As a possible solution, we suggest using Silica beads close to the drying plate to reduce humidity. We did, however, not test this approach as this protocol was not developed or tested in a humid climate.

Problem 1
Aberrant differentiation of iPSCs prior to starting neuronal differentiation (step 3).

Potential solution
Suboptimal iPSC culture protocol and/or issues with media components. Optimize iPSC handling according to commonly used practice and replace all media and consumables to troubleshoot issue. Scratch off differentiated cells under sterile conditions, e.g., using a hood equipped with a dissection microscope. Decrease time of PBS-EDTA incubation, e.g., to 4 min; most pluripotent cells will still detach while aberrantly differentiated cells will stay attached to the plate when rinsing with media.

Problem 2
Cells are not confluent or too dense after split into 12 well plate (step 3).

Potential solution
If cell density is too high, resulting in cell detachment, plate less cells; this is particularly important for very proliferative iPSC lines. If cell density is too low, resulting in holes in the cell layer, increase the number of plated cells during optimizations.
If visible clumping occurs in the days after plating into 12-wells, increase incubation time with Accutase and ensure cells are triturated to single cells before plating.

Problem 3
The cell line in use is difficult to differentiate into neuronal cells.

Potential solution
In general, the presented differentiation protocol may need to be optimized and adjusted for each new iPSC line. In particular, the different proliferation rates of different cell lines need to be taken into consideration regarding cell number and timing of different splits as described in the text. Beyond that, another option for difficult-to-differentiate cells is to adjust the neuronal induction step to minimize stress for the cells due to the sudden change from E8 to induction medium.
The neuronal induction step can be adjusted as follows.
use E6 -NI gradient on DIV0 and DIV1 starting with the split as described after step 3. On DIV0 perform split using 1 mL of E6-NI gradient medium-1 instead of NI. On DIV1 change medium E6-NI gradient medium-2. On DIV2 change medium to NI and use NI for all further feeds.

Problem 4
Cells do not attach after making spots or cell density becomes lower over time.

Potential solution
Quality of the cells before making the spots is crucial. Aberrant differentiation of iPSCs in the stem cell stage will impair the quality of the spots; it is therefore crucial to minimize aberrant differentiation before starting the differentiation to <5% as described in the ''Before You Begin''-section. It is important that the cell layer is homogeneous after induction: the cells should form a uniform layer without gaps and consistent density throughout the culture while on the 12-well plate, As described in step 4, it is important to ensure high nutrient and media supply, in particular towards the last days of induction in the 12-well plate; cells need to be fed daily with sufficient media. Cells should not peel off at the rim of the well or start dying. If this is the case repeatedly, cell number should be adjusted, and spots should be made earlier (i.e., DIV5 or DIV6). If dead cells are present or clumps form after collecting the cells from the 12-well plate, the spots and thus the entire differentiation are much more likely to fail. Low-quality drying of the POL-plates strongly affects attachment of spots to the plate; it is crucial that a uniform crystalline sheet without gaps is formed; if plates dry very fast (i.e., sheet is formed in <5-10 min) the airflow towards the plates should be reduced during drying to achieve drying times of 15-25 min. As mentioned in the limitations section, very humid conditions interfere with drying of the plates. Potential solutions might include adjustment of the airflow towards the plates or using Silica beads close to the drying plate to reduce humidity below 60%.

Problem 5
Spots become less dense over time or cells start detaching (step 11).

Potential solution
If spots become less dense, bFGF can be added to NM medium for the first feeds after NI (so starting from DIV10) to ensure the density of cells in the spot stays high. However, this should be limited to 2-3 consecutive days as otherwise other neuronal fates or non-neuronal cells may arise.

Problem 6
Cells are too sparse after Accutase splitting and/or rosettes were broken up during split (step 19).

Potential solution
NPCs should continue dividing after the split to re-form rosettes over the next days. This can be promoted by feeding with bFGF for 2 days as indicated in the protocol. If cells are still too sparse after 2 days, bFGF treatment can be continued for longer periods of time (e.g., until the next split). This may however also promote proliferation of aberrantly differentiated cells that were generated while the culture was not dense enough. We therefore recommend pausing bFGF treatment for at least 2 days between splits to promote cell death of aberrantly differentiated cells that may depend on bFGF signaling.

Problem 7
Neuronal differentiations produce not only cortical forebrain, but also other neuronal cell types.

Potential solution
5 mM XAV939, a WNT inhibitor, can be used on DIV0-2 (step 3/4) to promote cortical forebrain fate of the neurons. 13

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Dominik Paquet (Dominik.Paquet@med.uni-muenchen.de).

Materials availability
This study did not generate new unique reagents.

OPEN ACCESS
Data and code availability This study did not generate or analyze large datasets or code. Raw data for qPCR analysis is available from the corresponding author on request. The published article includes all other data generated or analyzed during this study.